Decontamination Of The BD FACSAria II - BD Biosciences

BD Biosciences 

July 2010 

Contents 

1 Abstract 

2 Introduction 

3 Objective 

3 Procedures 

5 Results and Conclusions 

5 Tips for Keeping Your Cytometer Free 

from Bacterial Contamination 

5 Tips for Performing a Contamination- 

Free Aseptic Sort 

5 References 

For Research Use Only. Not for use in diagnostic or therapeutic procedures. 

BD flow cytometers are Class I (1) laser products. 

Application Note 

Decontamination of the BD FACSAria II or BD FACSAria III System Using the Prepare for Aseptic 

Sort Procedure 

Decontamination of the BD FACSAria II or 

BD FACSAria III System Using the Prepare 

for Aseptic Sort Procedure 

Catherine A. McIntyre, Robert McCord, David Vrane 

BD Biosciences, San Jose, CA 


Abstract 

The ability to perform cell sorts that are free from bacterial contamination is 

vital for studies that rely on the subsequent culture of the sorted cells in an 

antibiotic-free environment. The presence of antibiotics can have a deleterious 

effect on cultured cell physiology and can also result in the generation of 

low levels of antibiotic-resistant strains of bacteria in the culture medium. The 

ability to decontaminate a cytometer in preparation for a sort is imperative, 

especially when there are multiple users and for studies that require a high 

level of sterility. The BD FACSAria II and III cell sorters were specifically 

designed with this in mind, and both provide several features that minimize the 

occurrence of contamination and facilitate the cleaning and decontamination of 

the fluidics system. The BD FACSAria II and III systems are research use only 

(RUO) instruments. 

In this proof of principle study, a BD FACSAria II flow cytometer was 

contaminated with an atypical level of bacteria to generate a “worst case” 

scenario that would not normally occur. These data demonstrate that the Prepare 

for Aseptic Sort procedure outlined in the BD FACSAria II User’s Guide is 

effective at decontaminating a BD FACSAria II flow cytometer contaminated 

with up to 9.8 x 10 5 CFU/mL of a mixture of different bacteria. The 

BD FACSAria II system remained bacterium free for at least four days after the 

Prepare for Aseptic Sort procedure was performed. The fluidics for the 

BD FACSAria III are identical to the BD FACSAria II, making the results of this 

study applicable to both instruments.


July 2010 




Introduction 

Bacterial contamination of cell products derived by sorting on a flow cytometer 

can compromise studies subsequently performed on the sorted cells. Many 

laboratories protect their sorted cell products from such contamination by 

using antibiotics in the medium for post-sort culture. Antibiotics can have a 

deleterious effect on the cells in culture and can lead to the presence of low levels 

of antibiotic-resistant bacterial contamination. The presence of these antibioticresistant 

bacteria might result in a depletion of essential nutrients and factors 

required by the cells in culture and the accumulation of bacterial metabolic 

waste products. These waste products might have unknown, unwanted, or 

toxic effects on cell metabolism or responses. 

A cell sorter is often used by several operators (eg, in a flow core lab) for the 

analysis and sorting of a variety of samples including, but not limited to, human- 

or animal-derived blood products and cultured cells. These samples might be 

contaminated with bacteria, viruses, or other pathogens that can potentially 

cause contamination of subsequent samples. In addition to samples, a cause of 

contamination is insufficient cleaning and maintenance of the cytometer and 

the use of fluids that have become contaminated (such as sheath fluid, deionized 

[DI] water, bleach, or ethanol). 

New fluidics design 

The BD FACSAria II and III flow cytometers were designed for easy 

cleaning and decontamination. When compared to the BD FACSAria, the 

BD FACSAria II and III instruments have a simplified sheath path with a reduced 

number of components, decreasing the fluid volume and the surface area available 

for contamination. The sheath tank is removable and autoclavable. Some of the 

tubing is now made from Teflon®, and the new fluidics system requires fewer 

valves. Valves are a manifold style (with less dead volume than the original style) 

and are very reliable. The sheath fluid path has a dedicated fluid line separate 

from the cleaning fluid line that is used only during cleaning and shutdown. 

Taken together, these new features reduce the opportunity for contamination 

and increase the efficacy of the cleaning and decontamination procedures. 

Prepare for Aseptic Sort procedure 

The Prepare for Aseptic Sort wizard in BD FACSDiva software has been 

designed to lead the BD FACSAria II and III operator through a decontamination 

of the fluidics system. When used in this context, the word aseptic does not 

imply that cells sorted on the BD FACSAria II or III systems will be free from 

all potentially infectious material, but only that this cleaning procedure can 

be used prior to sorting and can result in sorted cell suspensions free from 

bacterial contamination. When the operator initiates the procedure, the DI 

water, umbilical, and cytometer fluid pathways are rinsed, and the sample line 

is backflushed, followed by a soak in 10% bleach (0.5% sodium hypochlorite 

solution). This is followed by a rinse, backflush, and soak with DI water. Finally, 

the system is thoroughly flushed with ethanol to remove any residue. During the 

subsequent Fluidics Startup procedure, the umbilical and cytometer pathways 

are rinsed and the sample line is backflushed with sheath fluid, preparing the 

instrument for setup and sample introduction. 

The Prepare for Aseptic Sort procedure can be used after a known bacterial 

contamination has occurred, immediately prior to an aseptic sort, or as a 

routine maintenance procedure to minimize or prevent the occurrence of 

bacterial contamination in the cytometer’s fluidics system. 


BD flow cytometers are Class I (1) laser products.


July 2010 




Objective 

Page 3 

The objective of this application note is to demonstrate that in this proof of 

principle experiment the Prepare for Aseptic Sort procedure can remove high 

levels of bacterial contamination from the fluidics system of a BD FACSAria II 

flow cytometer. The fluidics for the BD FACSAria III are identical to the 

BD FACSAria II, making the results of this study applicable to both instruments. 

Procedures 

Table 1. Materials 

Material 

BD Falcon cell culture flask, 75 cm2 Pseudomonas aeruginosa (Schroeter) Migula 

Escherichia coli (Migula) Castellani and Chalmers 

Bacillus cereus Frankland and Frankland; deposited as 

Bacillus siamensis Siribaed 

Staphylococcus epidermidis (Winslow and Winslow) Evans 

Deionized (DI) water 

Clorox® Bleach Ultra 

Ethanol (70% solution, denatured, sterile) 

PBS (sterile concentrate, OmniPur®, 10X) 

Sheath filter 

Vendor 

BD 

ATCC 

ATCC 

ATCC 

ATCC 

BD in-house system 

VWR 

VWR 

VWR 

BD 

Part number 

353024 

35422 

25922 

7064 

55133 

N/A 

37001-060 

JTP004-3 

EM-6506 

331394 




July 2010 




Instrument setup 

The BD FACSAria II flow cytometer was prepared for this study by autoclaving 

the sheath tank and DI water containers (121°C, 15 psi, for 30 minutes). Freshly 

prepared sterile Dulbecco’s PBS solution was used to fill the sterile sheath tank. 

The three auxiliary cleaning fluid containers were then filled with sterile DI 

water, 70% ethanol, and 10% bleach (0.5% sodium hypochlorite solution in 

sterile DI water), respectively. The tank and containers were primed and the 

fluid filters bled. 

Contamination of the instrument with bacteria (day 0) 

A bacterial cocktail containing Pseudomonas aeruginosa, Escherichia coli, 

Bacillus cereus, and Staphylococcus epidermidis was created from individual 

bacterial cultures in log phase growth. The sheath tank and DI water container 

were inoculated with a sample of the bacterial cocktail. To contaminate the 

entire fluidics system, the sheath filter was removed from the sheath fluid line,* 

and the sheath tank was directly reconnected to the BD FACSAria II instrument. 

The cytometer was primed to move the contaminated fluids throughout the 

cytometer fluidics system. A Fluidics Startup was performed, the fluid stream 

started, a stable drop breakoff pattern established, and a sample line backflush 

performed to contaminate the sample line tubing. The instrument was then 

shut down with the bacterial contaminants in the fluidics system. 

Four days later (96 hours after inoculation) the BD FACSAria II cytometer was 

turned on, a fluidics startup performed, the fluid stream started, and a stable 

drop breakoff pattern established. One hundred milliliters of the sample stream 

was collected into a sterile BD Falcon cell culture flask for subsequent testing 

for the presence of bacteria. 

* In this experiment, the sheath filter was intentionally removed to allow the 

bacteria to circulate from the sheath tank throughout the entire fluidics system. 

Under normal circumstances, the 0.2-µm sheath filter present in the sheath 

fluid line prevents the spreading of any bacterial contamination that might be 

present in the sheath tank into the fluidics system. 

Decontamination of the instrument using the Prepare for Aseptic Sort 

procedure (day 4) 

The BD FACSAria II system was decontaminated using the Prepare for 

Aseptic Sort procedure as outlined in the BD FACSAria II User’s Guide. The 

sheath tank and DI water container were emptied and autoclaved, and the 

nozzle (with the o-ring in place) was soaked in a 70% ethanol solution. The 

Prepare for Aseptic Sort procedure was performed using the wizard within 

BD FACSDiva software, along with the hardware step of installing a new sheath 

filter. After completion of the procedure, a Fluidics Startup was performed, 

the nozzle insertion region of the cuvette was cleaned and dried using cotton 

swabs, and the cleaned nozzle (with the o-ring in place) reinserted. The fluid 

stream was started and a stable drop breakoff pattern established. 

Sample collection 

As before, 100 mL of the sample stream was collected into a sterile BD Falcon 

cell culture flask for subsequent testing for the presence of bacteria. The 

cytometer was shut down using the Fluidics Shutdown command. Samples of 

the sheath fluid were also collected on days 5, 6, 7, and 8. 




July 2010 




Testing of samples for bacterial contamination levels 

Page 5 

All samples were tested for the presence of bacterial contamination using 

standard QA testing procedures. 

Results and Conclusions 

Results are presented in Figure 1. In this study, a BD FACSAria II flow cytometer 

was contaminated with an atypical level of bacteria to generate a “worst case” 

scenario of bacterial contamination that would not normally occur. 

These data demonstrate that the Prepare for Aseptic Sort procedure outlined in 

the BD FACSAria II User’s Guide is an effective decontamination procedure 

that can be used with a BD FACSAria II flow cytometer contaminated with up 

to 9.8 x 10 5 CFU/mL of a mixture of different bacteria. In this experiment the 

BD FACSAria II system remained bacterium free for at least 4 days after the 

procedure was performed. 

The use of the Prepare for Aseptic Sort procedure does not guarantee that 

the fluidics system of a BD FACSAria II system will be free from bacteria but 

demonstrates that, in principle, a bacteria-free fluid path can be achieved. 

Customers should validate this procedure in their own laboratory. The 

BD FACSAria II system is a RUO instrument. The fluidics for the 

BD FACSAria III are identical to the BD FACSAria II, making the results of this 

study applicable to both instruments. 

Tips for Keeping Your Cytometer Free from Bacterial 

Contamination 

• Routinely maintain and clean your cytometer as outlined in the 

BD FACSAria II User’s Guide. 

• Most bacterial contamination comes from human skin cells and hair. Wear 

gloves when touching the fluidics system and tie back your hair. 

• If you use DI water from your facility’s central system, verify that it is serviced 

and sanitized on a regular basis. Consider using sterile water instead. 

• Do not “top up” the fluid containers and sheath tank. Instead, empty the 

residual fluid and start with fresh fluid each time. 

• Clean and autoclave the fluid containers and sheath tank on a regular basis. 

Tips for Performing a Contamination-Free Aseptic Sort 

• Use aseptic technique and sterile reagents (including antibodies) to prepare 

cells prior to sorting. 

• Clean the nozzle, clean the flow cell and deflection plates, and perform 

the Prepare for Aseptic Sort procedure immediately prior to using the 

cytometer for sorting. 

• Clean and autoclave the fluid tanks and containers and refill them with sterile 

reagents using aseptic technique. 

• Handle BD Cytometer Setup and Tracking beads, BD CompBeads, 

and BD FACS Accudrop beads using aseptic technique. Consider using fresh 

(unopened) bottles for an aseptic sort. 

• Collect sorted cells into sterile tubes and handle the cells using aseptic 

technique. 

References 

BD FACSAria II User’s Guide, Part Number 643245, Rev A, 2007 




July 2010 

Key: Fluid stream 




Figure 1. The Prepare for Aseptic Sort procedure decontaminates the BD FACSAria II system 

Day 0 

Day 4 

Days 5, 6, 7 & 8 

Log Phase Cultures 

P. aeruginosa E. coli B. cereus S. epidermis 

Startup & 

Backflush 

96 hrs 

Prepare for 

Aseptic Sort 

Procedure 

9 x 10 5 CFU/mL 

8 CFU/mL 

0 CFU/mL 




July 2010 


The BD FACSAria II and BD FACSAria III flow cytometers are For Research Use Only. 

Not for use in diagnostic or therapeutic procedures. 

BD flow cytometers are Class I (1) laser products. 

BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2010 BD 

23-10042-01 




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Decontamination Of The BD FACSAria II - BD Biosciences

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